The Cryptococcus wall: A different wall for a unique lifestyle

Introduction The life-threatening fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are differentiated from all other human fungal pathogens by the presence of a gelatinous capsule as well as an unusual underlying cell wall. These organisms have both beenAU : Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly: listed on the WHO list of fungal priority pathogens with higher disease burdens and unmet research and development needs, with C. neoformans at the top of the critical fungal priority group [1]. The aim of this review is to assess new insights into the unique attributes about Cryptococcus cell wall in relation to the pathogenic lifestyle of these important pathogens. Cryptococcus isolates have been classified into two species: C. neoformans (serotypes A, D, and AD) and C. gattii (serotypes B and C). Most studies on the Cryptococcus cell wall were performed using C. neoformans strains and, in many cases, without specifying the serotype used. For simplicity, here, I will only use the term “Cryptococcus” to indicate C. neoformans and C. gattii. Both the capsule and the cell wall contribute to the virulence properties of Cryptococcus and its ability to evade immune detection and killing (Table 1). The capsule and the cell wall are composed of different polysaccharides. While the capsule is mainly composed of glucuronoxylomannan and galactoxylomannan, the cell wall components include alphaand beta-glucans, chitin, chitosan, and mannoproteins (Table 1). The capsule is an essential virulence factor of Cryptococcus, and, as a consequence, its synthesis and function have been extensively studied (reviewed in [2,3]). In contrast, little attention has been paid to the Cryptococcus cell wall and its role in pathogenesis. Yet, the cell wall plays a crucial role in capsule synthesis and organisation [4,5], and defects in the Cryptococcus cell wall result into dramatic defects in cell division and morphology, increased sensitivity to stresses, and reduced virulence [6–9]. These observations strongly indicate that the cell wall also plays an important role in the biology of Cryptococcus and is a driver of Cryptococcus infection and disease.


Introduction
The life-threatening fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are differentiated from all other human fungal pathogens by the presence of a gelatinous capsule as well as an unusual underlying cell wall. These organisms have both beenAU : Pleaseconfirmthatallheadi listed on the WHO list of fungal priority pathogens with higher disease burdens and unmet research and development needs, with C. neoformans at the top of the critical fungal priority group [1]. The aim of this review is to assess new insights into the unique attributes about Cryptococcus cell wall in relation to the pathogenic lifestyle of these important pathogens.
Cryptococcus isolates have been classified into two species: C. neoformans (serotypes A, D, and AD) and C. gattii (serotypes B and C). Most studies on the Cryptococcus cell wall were performed using C. neoformans strains and, in many cases, without specifying the serotype used. For simplicity, here, I will only use the term "Cryptococcus" to indicate C. neoformans and C. gattii.
Both the capsule and the cell wall contribute to the virulence properties of Cryptococcus and its ability to evade immune detection and killing ( Table 1). The capsule and the cell wall are composed of different polysaccharides. While the capsule is mainly composed of glucuronoxylomannan and galactoxylomannan, the cell wall components include alpha-and beta-glucans, chitin, chitosan, and mannoproteins ( Table 1). The capsule is an essential virulence factor of Cryptococcus, and, as a consequence, its synthesis and function have been extensively studied (reviewed in [2,3]). In contrast, little attention has been paid to the Cryptococcus cell wall and its role in pathogenesis. Yet, the cell wall plays a crucial role in capsule synthesis and organisation [4,5], and defects in the Cryptococcus cell wall result into dramatic defects in cell division and morphology, increased sensitivity to stresses, and reduced virulence [6][7][8][9]. These observations strongly indicate that the cell wall also plays an important role in the biology of Cryptococcus and is a driver of Cryptococcus infection and disease.

Cryptococcus cell wall composition differs from that of other major human fungal pathogens
The Cryptococcus cell wall is mainly composed of glucans (α-1,3-glucan, β-1,3-glucan, and β-1,6-glucan), glycoproteins, chitin, and its deacelylated form chitosan (Table 1 and reviewed in [3]), but also contains melanin and lipids [10,11]. Although most fungal cell walls consist of similar polysaccharides, differences between Cryptococcus cell wall and the walls of other common fungal pathogens have been observed.

Chitosan
Compared to other major human fungal pathogens, Cryptococcus cells wall contains relatively high amounts of chitosan. In Cryptococcus, the wall chitosan content is 3 to 5 times higher than chitin during vegetative growth [13]. This is similar to the less clinically common zygomycete pathogens where 65% to 95% of the chitin is deacetylated [14]. Chitosan is also present in the ascospores of S. cerevisiae and the chlamydospores of Candida dubliniensis in small amounts, but absent in the vegetative cell wall of the yeast cells [15,16]. The cell wall of major pathogens Aspergillus fumigatus, C. albicans, and Pneumocystis jirovecii contain little or immeasurable chitosan. In Cryptococcus, chitosan is present in both in vitro-grown cells and cells isolated from infected mice [6,17], and chitosan deficiency has been associated with a reduced virulence [6]. Therefore, the Cryptococcus cell wall is particular in containing chitosan in both vegetative growth and in vivo, and chitosan is required for Cryptococcus pathogenesis.

The cell wall structure varies between Cryptococcus yeast cells of different sizes
The fungal cell wall is a dynamic and flexible structure that change significantly in composition during normal cell growth, environmental adaptation, or during morphological transitions. When grown in standard laboratory growth conditions, Cryptococcus cells appear as a homogenous population of 5 to 7 μm "normal-sized" yeasts [26]. In contrast, yeast cells extracted from infected tissues are of varying sizes and morphological characteristics [26,27]. This dynamic population includes greatly enlarged cells called "titan cells" (10 to 100 μm in diameter), "normal-sized" yeasts, and smaller cells (less than 4 μm of diameter) called titanides, seeder cells, and micro/drop cells [26][27][28][29]. Titan cells are so large that they may present challenges to efficient immune cell phagocytosis [26]. In addition to differences in cell sizes, these cell populations present differences in the structure of their cell wall. Titan cells have a significantly thicker cell wall (2 to 3 μm) than normal-sized cells (0.05 to 0.1 μm) [30] and have increased chitin and mannose contents [17,31]. Titanides, seeder, and micro/drop cells are small, and their cell wall structure also differs significantly from normal yeasts. Drop cells are round and have a thicker cell wall [28], while titanides are oval and have a thin cell wall [29].
The newly characterised seeder cell population is similar in size to titanides and have more exposed mannan than larger cells [27]. Therefore, the host immune system must be capable of recognising Cryptococcus yeast cells with significant differences in their size as well as their capsule and cell wall composition. However, the precise role of each morphotype in the immune recognition of Cryptococcus is not fully understood.

Both the capsule and the cell wall of Cryptococcus wall influence the host immune response
Because it is enveloped by the capsule, it is not clear how the cell wall actively engages in immune activation. However, it is clear that the wall also contributes to immune recognition and the immune response to this fungus (Table 1; [17,32]). Chitin and chitosan have been associated with nonprotective immune responses [17,18], although they are in the inner layer of the cell wall and covered by other wall components and by the capsule. This is problematic in understanding how the interaction between chitin/chitosan and immune cells occurs, or whether it is triggered by intact cells or by cell wall fragments that are shed by the yeast cell.
Cell wall β-1,3-glucans have been detected in the cerebrospinal fluid and serum of HIV+ patients with Cryptococcus meningitis and were associated with pro-inflammatory chemokine responses [33]. In addition, mannoproteins recovered from the Cryptococcus culture supernatant stimulate T-cell immune responses [22,23]. Cryptococcus releases capsule polysaccharides into the extracellular space during infection and in in vitro culture, and the shed polysaccharides modulate the host immune responses [32]. Similarly, cell wall components may be shed and interact with the host cells indirectly. It is not known whether cell wall components, other than β-1,3-glucans and mannoproteins, are also shed during Cryptococcus infection and contribute to immune stimulation.

The cell wall and limitations in the use of antifungal drugs
Echinocandin antifungal drugs (caspofungin, anidulafungin, and micafungin) inhibit β-1,3-glucan synthesis, resulting in the disruption of cell wall integrity and, ultimately, fungal cell death [34]. Although echinocandins are active against most Candida and Aspergillus species, they are largely ineffective against Cryptococcus in vivo [34,35]. This is surprising in so far as the FKS1 gene that encodes for β-1,3-glucan synthase is essential in Cryptococcus [20], and this enzyme is sensitive to echinocandins in vitro [36]. The mechanisms behind the resistance to echinocandins are not well understood.
In comparison to other yeasts, Cryptococcus cell wall contains more β-1,6-glucans than β-1,3-glucans. Could this difference in β-glucans impact the resistance of Cryptococcus to echinocandins? A previous study showed that treating Cryptococcus with caspofungin resulted in the reduction of both β-1,3-glucans and β-1,6-glucans, and concluded that inhibition of β-1,6-glucans may be an additional mechanism of action of pneumocandin [37]. Therefore, increased β-1,6-glucans in Cryptococcus cell wall does not explain its resistance to echinocandins.
Another possibility is that in vivo cell adaptations such as the capsule and the thick cell wall could prevent access of echinocandins to their target enzyme. Studies using acapsular and melanin-deficient mutants found that the capsule and melanin were not required for the caspofungin resistance [38]. However, lipid flippase defects in the cell membrane were associated with higher caspofungin penetration into the cell and increased caspofungin susceptibility [38]. In response to caspofungin, Cryptococcus increased its chitin and chitosan contents [39], a compensatory mechanism similar that observed in Candida species and A. fumigatus [40]. Therefore, both the cell wall and plasma membrane integrity may play a role in Cryptococcus resistance to echinocandins.
Chitin synthase inhibitors have been investigated as antifungal drugs and some (e.g. Nikkomycins) have shown in vitro and in vivo activity against fungal pathogens such as Coccidioides and Blastomyces species [41]. These chitin synthase inhibitors do not have a strong activity against Cryptococcus, and currently, there is no chitin synthase inhibitor in clinical use.

Concluding remarks
The fungal wall is an ideal target for the development of new antifungal drugs. Cryptococcus cell wall differs in design and composition from that of other major human fungal pathogens. Although substantial work is still needed to fully understand the role of each wall component in immune recognition/evasion and/or antifungal drug resistance, information presented here emphasizes that the cell wall is a key player in Cryptococcus pathogenicity and could be a potential target of new anti-Cryptococcus drugs.